Dr Aleksandar Ivetic BSc (Hons) ARCS PhD
King's College London
James Black Centre
125 Coldharbour Lane
London SE5 9NU
Dr Ivetic was appointed Senior Lecturer in Cardiovascular Biology in April 2009 and is Principal Investigator of the Membrane/Cytoskeleton Signalling Group. Dr Ivetic graduated from Imperial College London with a BSc (Hons) in 1993 in Biochemistry and additionally became an Associate of the Royal College of Science (ARCS). He then moved to the Marie Curie Research Institute (Oxted, Surrey) in 1994 to undertake a PhD (awarded by the Institute of Cancer Research, University of London) in Biochemistry to understand the role of the Cell Cycle in regulating DNA replication.
His first post-doctoral position was held at the National Institute for Medical Research (NIMR, Mill Hill, London) where he applied his expertise in protein biochemistry to the field of leukocyte trafficking. He then moved to the Ludwig Institute for Cancer Research (UCL branch, London) for his second postdoctoral position, where he gained extensive knowledge in cell biology, with a main focus on leukocytes, fibroblasts and endothelial cells. He subsequently moved to Imperial College London (National Heart and Lung Institute) in July 2005 as part of a Wellcome Trust Research Career Development Award, which enabled him to continue his interests in leukocyte trafficking, and develop skills to analyse leukocyte adhesion under flow conditions.
Leukocyte trafficking in cardiovascular disease – the big goal
Inflammatory cells (such as lymphocytes, neutrophils and monocytes) are known to drive the progression of many chronic and acute cardiovascular diseases such as atherosclerosis, myocardial injury induced by ischemia and cardiac allograft rejection. Therefore, understanding the detailed molecular mechanisms by which leukocytes are recruited to sites of tissue injury within the cardiovascular system will provide the potential to develop therapeutic strategies to curb acute and chronic inflammatory disorders that plague tissues and organs during disease progression.
The Membrane/Cytoskeleton Signalling Group
Our primary research aim is to understand the molecular mechanisms that facilitate leukocyte adhesion to the luminal walls of blood vessels. Binding of leukocytes to the vessel wall is an absolute requirement for successful passage out of the vasculature and in to the surrounding tissue. This process is embodied by the “leukocyte multi-step adhesion cascade” (Figures 1 & 2), which is crudely broken down in to:
Initial capture (or tethering)
A number of different cell adhesion molecules on the surface of leukocytes are responsible for mediating tethering and rolling. One such molecule is called L-selectin, which is presented on the tips of finger-like projections called microvilli These microvilli provide L-selectin with a biological advantage to recognise its ligand over other cell adhesion molecules present on the plasma membrane. The cortical actin-based cytoskeleton is a key component in the formation of microvilli and is therefore though to play a major role in ensuring the correct positioning of L-selectin to microvilli.
Figure 1 - Simplified overview of the multi-step leukocyte adhesion cascade
(Courtesy of Marouan Zarrouk and Rufus Ho). A local inflammatory insult leads to signalling events that stimulate endothelial cells within the local vicinity to up-regulate cell adhesion molecules that promote leukocyte recruitment. Leukocytes come in initial contact with endothelial cells by making sub-second adhesive contacts (tethers) with the underlying endothelium. Tethering is translated in to rolling in the presence of sufficient ligand. The weak interaction between leukocytes and endothelial cells, in combination with blood flow, causes leukocytes to roll (like tumbleweed) along the endothelial cells. Engagement with chemokines is classically thought to promote arrest against blood flow, although several reports suggest that this can occur independently of chemokines stimulation (at least in vitro). Leukocytes finally exit the vasculature by transmigrating either through or between endothelial cells.
Figure 2 - Neutrophil transmigration through an endothelial cell monolayer
Neutrophils are perfused over TNF-activated human umbilical vein endothelial cells (HUVECs) and tracked over time by timelapse brightfield microscopy. Note that adherent neutrophils on top of HUVECs are “phase bright” and transmigrated neutrophils appear “phase dark”. Note also that dramatic changes in cell shape also occur during the transmigration phase.
We recently discovered the ezrin-radixin-moesin (ERM) family of proteins as binding partners of the short, 17 amino acid, cytoplasmic tail of L-selectin. ERMs essentially link the cortical actin cytoskeleton with the plasma membrane by generating direct interactions via their C- and N-termini (Figure 3). Interestingly, overexpression of cell adhesion molecules that bind ERM (such as L-selectin) can promote the formation of microvilli, and knocking down expression of ERMs can lead to the loss of microvilli. We have shown that abrogating L-selectin/ERM interaction reduces microvillar positioning of L-selectin, which in turn affects leukocyte tethering to immobilised ligand under conditions of flow (using in vitro flow chamber assays). Collectively, these observations suggest an inter-dependent relationship between L-selectin, microvilli and ERMs, the mechanism of which is poorly understood.
We have taken further steps to understand how such a short cytoplasmic tail can accommodate the binding of more than one partner and how this might be involved in mediating signal transduction during adhesion (Figure 4). Indeed, others have shown that clustering of L-selectin (which is thought to occur during L-selectin-dependent tethering and rolling) can promote mobilisation of the chemokine receptor, CXCR4, to the plasma membrane and (β1 and β2) integrin activation. Both of these events are necessary for progression through the adhesion cascade, and signalling through L-selectin is therefore though to be involved in mediating the transition from rolling to arrest – either independently of, or in concert with, chemokines.
Finally, surface levels of L-selectin are regulated by ectodomain shedding, which has been shown to regulate the extent to which leukocytes are recruited to sites of inflammation. The enzyme that cleaves L-selectin at the membrane-proximal extracellular domain is called TNF-α converting enzyme (TACE/ADAM17). We have a strong interest in understanding how the cytoplasmic tails of L-selectin and TACE are regulated to promote ectodomain shedding. Insights in to these processes could help us understand how shedding regulates adhesion and signalling during recruitment.
Figure 3 - Schematic model of how ERM proteins are regulated
In their inactive states, the N- (red clover-leaf shape) and C- (green oval) termini interact with one another to form either an auto-inhibited “closed” conformation. Others have reported that ERMs can bind in an anti-parallel fashion. In either case, it is the interaction between N- and C-termini that masks binding sites for interaction with other proteins. Phosphorylation of a conserved threonine residue results in the opening of the closed conformation. This enables the N- and C-termini to bind to the tails of cell adhesion molecules (such as L-selectin) and the cortical actin cytoskeleton, respectively. ERMs can also flip open by binding to PIP2, a phospholipid found in the inner leaflet of the plasma membrane which is involved in recruiting many other cytoskeleton proteins. The relative activities of the RhoGTPases RhoA and Rac have been shown to have influences on the activity of ERMs, but these effects may be cell-type-specific. Image adapted from reference 6.
Figure 4 - Possible signalling mechanisms during cell rolling
(A) Molecular modelling reveals that the short cytoplasmic tail of L-selectin can accommodate the binding of both calmodulin and ERM (e.g. N-terminal moesin, or FERM). We have recently shown, using fluorescence lifetime imaging microscopy (FLIM) to monitor fluorescence resonance energy transfer (FRET), that calmodulin and ERM interact with one another in intact cells (B), which supports our previous in vitro and in silico findings (see reference 1 for more information). This implies that remodelling of the extracellular domains of L-selectin (through ligand binding) leads to changes in how L-selectin complexes with its cytosolic binding partners (C). Such remodelling events could be involved in signalling during cell tethering and rolling and therefore facilitate the transition from rolling to arrest.
In vitro flow assays (fluorescence timelapse video microscopy)
Mr Ross King
Ms Abigail Newe
Ms Karolina Rzeniewicz
Ms Hannah Tomlins
From left to right: Ms Abigail Newe, Dr Angela Rey Gallardo, Ms Karolina Rzeniewicz, Ms Hannah Tomlins
Rzeniewicz K, Newe A, Rey Gallardo A, Davies J, Holt MR, Patel A, Charras GT, Stramer B, Molenaar C, Tedder TF, Parsons M, Ivetic A. L-selectin shedding is activated specifically within transmigrating pseudopods of monocytes to regulate cell polarity in vitro. Proc Natl Acad Sci U S A. 2015 Mar 9. pii: 201417100